The foundation for this project is a robust description of erythropoiesis and the human erythroid transcriptome. We have begun the systematic characterization of cellular events that are manifest through transcription in primary human erythroblasts. Our goal is to utilize the erythroid transcriptome in order to understand and therapeutically manipulate erythroid cellular events in the context of disease. Erythroid diseases encompass a broad range of anemias, hemoglobinopathies and malarial syndromes. Accomplishments achieved during the last year: 1. Hembase: a description of the human erythroid transcriptome. Previously, we reported the sequencing of human erythroblast libraries to generated an informatic database describing the erythroid transcriptome. That database, called Hembase (http://hembase.niddk.nih.gov/), was comprised of homology comparisons from our Expressed Sequence Tag (EST) collection with sequences contained within other publicly available databases. During the last year, Hembase was reformatted and redesigned largely due to the completion of the human genome map. The new Hembase was launched on the Internet in August of 2003. The current version of Hembase now consists of 16,000 searchable files with direct links to the primary sequence data as well as specific links to three major human genome browsers currently in the public domain. Conceptually, a genome portal format was created to provide search-current information to the users rather than static information that may no longer be current at the time of each query. Hembase is the first human genome portal placed in the public domain. 2. Studies of fetal hemoglobin modulation. In recent decades, models used to approach reactivation of the fetal hemoglobin genes were based on the assumption that erythroblasts normally undergo a switch from fetal-type to adult-type hemoglobins during the differentiation process. Information gathered in Hembase (above) led us to hypothesize and report in 2002 that fetal hemoglobin normally accumulates in adult human cells with a pattern similar to that of adult hemoglobin. Importantly, we also determined that the pattern of hemoglobin accumulation during adult erythropoiesis is directly related to the proliferation of the cells. That new model for hemoglobin production led us to novel hypotheses that regulation of signal transduction cascades and growth among fully committed erythroid cells may be used to increase levels of fetal hemoglobin. During the last year, this signaling hypothesis has become a major focus of my laboratory. First, we developed a standard experimental assay of cultured human erythroid progenitor cells obtained from normal volunteers to identify cytokines capable of modulating erythroid growth and fetal hemoglobin (HbF) production during adult erythropoiesis. As part of that initial study, twelve erythroid growth-promoting cytokines were screened, and stem cell factor (SCF) resulted in the most significant increase in cell proliferation and HbF content. To further investigate the hemoglobin-modulating effects of SCF, we next determined that expression of the SCF receptor, CD117, among maturing erythroblasts peaked at the proerythroblast stage of differentiation. SCF-related increases in the HbF/HbA ratio were then correlated with the expression pattern of CD117 to demonstrate the developmental stage at which SCF has its major effects on HbF. A significant SCF-mediated increase in HbF was also measured using progenitors derived from cord blood. Conceptually, these results suggest that new therapies aimed at the manipulation of erythroblast signaling cascades may be developed for the treatment of sickle-cell diseases and beta-thalassemias. 3. Identification of Nix as an erythroid growth regulator. Studies aimed toward a more complete understanding of growth-inhibition during terminal cellular differentiation remain an active area of translational research aimed toward the treatment of leukemias and other growth-related diseases. Ex vivo cultures of erythroid cells provide one of the only experimental system for the study of terminal cellular differentiation using primary cells from human donors. Relatively little in known about the mechanism(s) that inhibit growth during terminal erythroid differentiation. We previously determined that erythroid gene patterning is useful for the identification of those elements that are dysregulated in patients with myelodysplastic syndromes. We have more recently begun to screen the erythroid transcriptome for growth inhibiting genes that are normally expressed during terminal erythroid differentiation. During the last year, we screened Hembase and determined that the hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during terminal erythroid differentiation. While increased expression of Nix in the context of apoptotic cardiomyopathies is known, our discovery of its expression in hematopoietic cells is novel. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than in reticulocytes. Polymerase chain reaction-based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we proposed that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation. We believe this and other related gene discovery projects should significantly enhance efforts aimed toward understanding the regulation of growth during terminal cellular differentiation. 4. Collaborative studies involving erythroid cell flow cytometry. Over the last 8 years, we have developed considerable expertise in the area of erythroid flow cytometry based upon our almost daily requirement of differentiation analyses or highly purified populations. Based upon those expertise, we have also engaged in primary and collaborative studies involving flow cytometry of erythroid cells with other intramural researchers. In addition, we have provided general flow cytometric assistance (analyses and cell sorting) to several other NIDDK intramural laboratories. During 2003, two collaborative projects of this type were completed and reported. In the first, flow cytometry was compared with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins. With colleagues from the NIH Department of Transfusion Medicine, RBCs from 20 patients with reactive tube direct antiglobulin tests were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. We determined that flow cytometry is more sensitive than the tube assay for the detection of RBC-associated C3d. These results suggest that clinical flow cytometry may be useful for the diagnosis of immune-mediated hemolysis at levels below the detection limits of standard assays. The second project (collaboration with Dr. Alan Schechter) involved the use of flow cytometry for analysis of erythroid cells treated with hydroxyurea.